Molecular Genetics Flashcards
Name 2 X-Linked Recessive disorders
Duchenne/Becker Muscular Dystrophy
SBMA
Androgen Insensitivity Syndrome
Name 2 X-Linked Dominant disorders
X-linked hypophosphatemia
X-linked Alport Syndrome
Rett Syndrome
Name three clinical features associated with CF
Chronic coughing and wheezing
Failure to thrive
Pancreatic insufficiency
Name the most common CF mutation
p.Phe508del - 75%
Describe the function of CFTR (7q31.2)
Cyclic AMP-activated chloride channel located in the plasma membrane of secretory eithelial cells
How many types of SMA are there?
6 - Prenatal, type 1, Type 2, Type 3, Type 4 and atypical SMA
Describe 3 characteristic features of SMA
Progressive proximal symmetrical limb and trunk muscle weakness,
Intercostal muscle weakness,
Fine tremor
Facial weakness
What is the most common type of SMA? Describe
Type 1 - profound hypotonia, symmetrical flaccid paralysis, progressive, death at early age
Give me some molecular facts about SMA
SMN1 and pseudogene SMN2
4% population have 2 copies of SMN1 on one chrm (range 0-5)
95-98% SMA homozygous for deletion of at least exon 7
2-5% are compound heterozygotes for del and pathogenic inactivating mutn
How do we interpret SMA carrier testing?
Bayes calculation
Briefly describe CRISPR-Cas9
Gene editing - Cas9 protein complex containing specific sequence of RNA - once complimentary sequence is identified the DNA is cut and released into the cell. Cellular DNA repair mechanisms repair the break - but this is prone to error. By introducing templates of ‘corrected’ DNA - these specific sequences can be incorporated to replace mutant alleles
What is the clinical significance of 36-39 CAG repeats in Huntington Disease?
Reduced penetrance - may or may not be affected
Above what number of CAG repeats would you see fully penetrant development of Huntington Disease?
40 CAG repeats
What is the significance of 27-35 CAG repeats in Huntington Disease?
Intermediate allele - will not cause disease but may expand to cause disease if paternally transmitted
Name 3 disorders associated with FMR1
Fragile X, FXTAS and POI
How is Fragile X caused?
Expansion of unstable 5’ UTR CGG repeat to >200 repeats, causing gene silencing
Give 3 clinical features of Fragile X in males
moderate/severe intellectual and social impairment, characteristic facial features, joint laxity, macro-orchidism
Give the clinical presentation of Fragile X in females
Variable phenotype - apparently normal approx 50%) through to mild/moderate mental and social impairment
What is the significance of repeat size of 55-200 in FMR1?
Premutation - may expand in maternal line to cause FraX in future generations. Patients may develop FXTAS or POI
What is the proposed disease mechanism for FXTAS/POI
NOT the same as FraX - ?toxic gain of function?
What is the location of FMR1?
Xq27.3
What is the role of FMRP protein?
RNA binding protein, present in many tissues including brain, ovaries and testes - thought to act as a shuttle within cells by transporting mRNA from nucleus to areas of cell where proteins assembled. Also helps to control when the instructions in these mRNA molecules are used to build proteins.
Name 2 NGS platforms
Agilent Sure Select, ThermoFisher Ion Torrent, Illumina HiSeq/MiSeq SBS
What are the mutation ranges for SBMA?
Normal - 34 CAG repeats or less Questionable - 35 CAG repeats Reduced penetrance - 36-37 CAG repeats Affected fully penetrant - 38 CAG repeats or greater Sequence any 35-37 repeats
Name 3 CAG expansion disorders
Huntingtons
SBMA
SCAs - 7 of them
Name 2 CGG expansion disorders
Fragile X
FXTAS
Name 3 CTG expansion disorders
Myotonic Dystrophy 1
Huntington Like Disease 2
SCA 8
Name a GAA expansion disorder
Friedreich Ataxia
Name a CCTG expansion disorder
Myotonic Dystrophy 2
What additional features might you see in a juvenile HD patient with a large expansion?
Bradykinesia
Dystonia
What factors can contribute to pathogenic mechanism of repeat expansion diseases
Sequence of repeat Size of repeat Location of repeat within gene Whether repeat encodes RNA or protein Function of repeat-containing gene Extent of meiosis and somatic instability
What causes Myotonic Dystrophy 1?
CTG expansion in the 3’UTR of the DMPK gene
What causes Myotonic Dystrophy 2?
CCTG repeat in intron 1 of the CNBP gene
What are some clinical features of DM1?
Progressive weakness and myotonia Cataracts Cardiac arrhythmias Endocrinopathy Cognitive impairment
Name 7 methods for detecting UPD
MS-PCR MS-Melt Curve Analysis MS-Pyrosequencing Microsatellite analysis MS-MLPA SNP Arrays WGS/WES - trios especially powerful
Name 3 methods of detecting UPD that rely on bisulphite conversion
MS-PCR
MS-Melt Curve Analysis
MS-Pyrosequencing
What are the pros and cons of using microsatellite analysis for UPD detection?
Pros: Will distinguish whole/partial UPD and Hetero/Isodisomy, relatively cheap, no prior conversion step, reliable method
Cons: Need parental samples and informative markers
Describe MS-MLPA
2 tubes, 1 for CNV, 1 for methylation specific enzyme digest. Methylated DNA won’t digest, and then will amplify during MLPA.
Semi quantitative, no parental bloods need, can distinguish UPD from deletion from a small amount of DNA.
No need for parental samples, can’t distinguish UPD and mutation in imprinting centre
How can you use WGS/WES for detection of UPD?
Trio analysis - bioinformatic pipelines will check for identity by looking for biparental inheritance
Can detect mosaics and can distinguish full range of del/UPD/IC defect
Expensive and time consuming, requires parental samples
What are the pros and cons of using bisulphite conversion for UPD testing?
Allows for analysis without parental samples, bisulphite conversion kits readily available, cheap and effective
Won’t distinguish segmental/whole UPD, or del from UPD (MS-PCR)
Describe Class 1 CFTR mutations
Nonsense, most frameshift mutns and large del’s create premature stop codons causing defective protein synthesis and no CFTR protein expressed. Severe phenotype, W128X, R553X, G542X. Treatments - readthrough drugs - Ataluren phase 3 trials
Describe Class 2 CFTR mutations
Some missense mutns and in frame del’s disrupt CFTR protein folding and trafficking to the surface. F506del, N1303K. Treatments: correctors to promote folding - Lumacafter
Describe Class 3 CFTR mutations
Some missense mutns result in substitution of aa’s, disrupting regulation of CFTR channel, which no longer opens in response to channel agonists. G551D, G551S, G1349D. Treatment Ivacafter being trialled.
Describe Class 4 CFTR mutations
Some missense mutations result in changes to CFTR protein structure that forms the pore of the channel which can restrict the movement of Cl- ions through the channel - conductance defect. Eg R117H, R334W, R347P. Ivacafter being trialled.
Describe Class 5 CFTR mutations
Some missense mutations result in alternative splicing that disrupts mRNA processing - extremely reduced amounts of normal CFTR protein are synthesised, less protein at cell surface. 2789+5G>A, A455E. Treatments are focussed on compounds that enhance CFTR retention/anchoring
Describe Class 6 CFTR mutations
Different types of mutations increase the turnover of CFTR protein at the cell surface, quickly removed and degraded by cell machinery. Egs include Rescued F508del, 120del23, N287Y, 4326delTC, 4279insA
How many classes of CFTR mutations are there?
6
